Quantitation of functional groups on solid supports

ABSTRACT

Processes for quantifying an amount of functional groups immobilized on a solid support are described herein. The processes allow for determining whether sufficient functional groups are provided on a solid support for the attachment of a first binding pair member for the detection of a target analyte.

FIELD OF THE INVENTION

The present invention relates to the field of diagnostic testing, andmore particularly to methods for quantifying functional groupsimmobilized on a solid support.

BACKGROUND OF THE INVENTION

In the fields of medicine and clinical chemistry, many studies anddeterminations of physiologically reactive species or analytes arecarried out by taking advantage of the interaction between specificbinding pair members. For example, the target analyte in a patientsample may itself be one member of a specific binding pair, which may bedetected by employing a corresponding member of the specific bindingpair immobilized on a solid support. In this case, the immobilizedbinding pair member may be an antigen for the detection of a targetantibody in a sample, for example. Various solid support materials havebeen developed for these applications and require various bondingtechniques to immobilize the specific binding pair member on the solidsupport.

For example, paramagnetic particles (PMP) are known solid supportmaterials which may be functionalized with functional groups such asamine functional groups. In some instances, these functional groups maybe selected so as to bond with a linking group (e.g., glutaraldehyde),which bonds to a binding pair member. The binding pair member may beselective for a target analyte in a sample, which is typically a secondmember of the binding pair as mentioned above. In immobilizing thebinding pair member on the solid support, it may be necessary to ensurea proper amount of the functional groups are available for direct orindirect attachment of the binding pair member. Too few availablefunctional groups, for example, may result in too few attachment siteswhen the binding pair member is added. This may result in a solidsupport incapable of accurately determining an amount of a targetanalyte at certain concentrations, particularly at higher concentrationsin the detectable range of the target analyte. On the other hand,providing greater concentrations of the functional groups for theimmobilization of the specific binding member may substantially increaseproduction costs without any added benefit.

Accordingly, there is a need to determine and/or confirm the number offunctional groups, directly or indirectly, present on a solid support toin order to optimally immobilize the specific binding pair members tothe solid support. While there are known methods for quantitativelydetermine an amount of functional groups in solution, such methods aretypically unsuitable for solid supports since colored or fluorescentreaction products formed in such quantification methods typically bindto the solid support, thereby rendering detection and quantitationdifficult and/or impossible.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention are explained in the following description inview of the drawings that show:

FIG. 1 illustrates a prior art solid support having a first binding pairmember immobilized thereon for detection of a second complementarybinding pair member in a sample in accordance with an aspect of thepresent invention.

FIG. 2 illustrates a solid support having surface functional groups anda selective compound bound to the surface functional groups inaccordance with an aspect of the present invention.

FIG. 3 illustrates a supernatant comprising a selective compound boundto an indicator in accordance with an aspect of the present invention.

FIG. 4 illustrates a solid support having a linking agent and aselective compound bound to a linking agent in accordance with an aspectof the present invention.

FIG. 5 illustrates a method for determining a number of functionalgroups on a solid support in accordance with an aspect of the presentinvention.

FIG. 6 is a graph showing concentrations of propionaldehyde standardsvs. absorbance in accordance with an aspect of the present invention.

FIG. 7 is a graph showing absorbance of sample supernatants from timedependence studies in accordance with another aspect of the presentinvention.

FIG. 8 is a graph showing concentrations of propionaldehyde standardsvs. absorbance in accordance with another aspect of the presentinvention.

FIG. 9 is a graph showing time dependence of Schiff-base formation foraldehyde quantitation in accordance with an aspect of the presentinvention.

FIG. 10 is a graph showing concentrations of propylamine standards vs.absorbance in accordance with an aspect of the present invention.

FIG. 11 is an effect plot indicating % glutaraldehyde as a main factorof aldehyde group formation in accordance with an aspect of the presentinvention.

FIG. 12 is an interaction plot showing PMP mg/mL intersecting with %vessel occupancy in accordance with another aspect of the presentinvention.

FIG. 13 is a Pareto Chart illustrating B (% glutaraldehyde) as a maineffect of aldehyde group formation in accordance with another aspect ofthe present invention.

FIG. 14 illustrates optimal PMP concentration, glutaraldehydeconcentration, and vessel occupancy to optimize aldehyde loading on PMPin accordance with another aspect of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there areprovided processes for quantifying an amount of functional groupsimmobilized on a solid support. In certain aspects, the processes allowfor the determination of an optimal number of functional groups to bedisposed on a solid support during manufacture of a solid supportproduct. In this way, waste may be eliminated and/or reduced, andperformance of the solid support may be improved upon. For example, byquantifying the number of functional groups available on a solidsupport, it can be assured that adequate functional groups are availablefor direct or indirect attachment of a first member of a specificbinding pair. Thereafter, like solid supports can be manufactured withan optimized loading of the specific binding pair member. In certainembodiments, the processes described herein thus allow for thequantitation of available functional groups prior to the direct orindirect attachment of one member of a specific binding pair provided onthe solid support.

As used herein, the term “about” refers to a value that is ±10% of thestated value.

As used herein, the terms “attachment,” “binding,” “immobilized,”“linking,” or the like are understood not to be limited to covalentbonding, and may include any type of attraction, affinity,conformational selection, induced fit, or bonding between two or moremolecules.

As used herein, by the phrase “effective amount,” it is meant an amountof material suitable for bringing about an intended result.

As used herein, the term “sample” includes any sample having orsuspected of having the target analyte, and may be a biological ornon-biological sample.

As used herein, the term “subject” refers to any human or non-humanmammal.

Referring to FIG. 1, there is shown an exemplary solid support 10 havingsurface functional groups 12 on a surface thereof. In the embodimentshown, there is also provided a linking agent 14 for the immobilizationof a first binding pair member 16 to the solid support 10. It isunderstood however that the present invention does not require such alinking agent 14, and that the first binding pair member 16 may insteadbe bonded directly to the surface functional groups 12. The firstbinding pair member 16 may be suitable for conjugation with a secondbinding pair member 18 in a sample 20 as shown. The sample 20 may be astandard sample, or a sample (e.g., biological sample) suspected ofhaving the second binding pair member 18. As such, the second bindingpair member 18 may be considered a target analyte for the solid support10.

The solid support 10 may be comprised of an organic or inorganic waterinsoluble and impermeable material, which may also be transparent orpartially transparent. In addition, the solid support 10 may be in theform of a bead, particle, fiber, film, membrane, tube, well, a strip,rod, a planar surface such as a plate, and the like. Depending on thetype of assay for which the solid support 10 is intended to be used, thesolid support 10 may or may not be suspendable in the medium in which itis employed. Examples of suspendable supports include but are notlimited to polymeric materials such as latex, lipid bilayers orliposomes, oil droplets, cells and hydrogels, magnetic particles, andthe like. Other solid support materials include polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), etc.; either used alone orin conjunction with other materials.

In certain embodiments, the solid support 10 comprises one or more solidparticles. When in the form of particles, the particles may have anaverage diameter of at least about 0.02 microns to about 100 microns,for example. In particular embodiments, solid particles may have anaverage diameter from about 0.05 microns to about 20 microns, or fromabout 0.3 microns to about 10 microns. In addition, the particles mayhave a surface area range of about 10 to about 100 m²/g, and in someembodiments the particles may have a surface area in the range of about10 to about 60 m²/g. The particle may be organic or inorganic, swellableor non-swellable, porous or non-porous, and may have a densityapproximating water, generally from about 0.7 g/mL to about 1.5 g/mL,and composed of material that can be transparent, partially transparent,or opaque.

The particles may have a regular or irregular shape. They may be, forexample, spheres, spheroids or spheres possessing cavities or pores. Theparticles may comprise several layers, such as what are termed core andshell particles, having a core and one or more enveloping layers. Incertain embodiments, the particles may be formed from biologicalmaterials such as cells and microorganisms, e.g., erythrocytes,leukocytes, lymphocytes, hybridomas, streptococcus, Staphylococcusaureus, E. coli, viruses, and the like. The particles can also beparticles comprised of organic and inorganic polymers, liposomes, latexparticles, magnetic or non-magnetic particles, phospholipid vesicles,chylomicrons, lipoproteins, dye crystals, metal sols, silica particles,glass particles, magnetic particles, oil drops, lipid particles, dextranand protein aggregates and the like.

In certain embodiments, the particles comprise nanoparticles and/ormicroparticles. Such particles may have an approximate diameter of atleast about 20 nm and not more than about 20 microns, or between 40 nmand 10 microns, or between 0.1 and 10 microns, or between 0.1 and 5microns, or between 0.15 and 2 microns. In particular embodiments, themicroparticles may be particles that are suspended in aqueous solutions.

The particles may comprise polymer particles that can be dispersedand/or suspended within an aqueous solution. In certain embodiments, theparticles may be readily dispersible in an aqueous medium, and can beadsorptive or functionalizable as will be explained below so as topermit conjugation to a member of a specific binding pair, eitherdirectly or indirectly (through a linking agent). The particles may alsobe derived from naturally occurring materials, naturally occurringmaterials that are synthetically modified, and synthetic materials.Exemplary organic polymers include polysaccharides, particularlycross-linked polysaccharides, such as agarose, which is available asSEPHAROSE® (Cytiva, Marlborough, Mass.), dextran, available as SEPHADEX®and SEPHACRYL® (GE Healthcare, Chicago, Ill.), cellulose, starch, andthe like; addition polymers, such as polystyrene, polyvinyl alcohol,homopolymers and copolymers of derivatives of acrylate and methacrylate,such as esters and amides having free hydroxyl functionalities, and thelike.

In certain embodiments, the solid support 10 comprises one or moremagnetic particles such as paramagnetic particles. When the particlesare magnetic, the magnetic material contained in the particles may beany magnetic material susceptible to attraction by a permanent magnet oran electromagnet. Examples of such magnetic materials include magneticiron oxides, magnetic chromium dioxides (CrO₂), MnFeO₄, ZnFeO₄, CoFe₂O₄,and similar magnetic materials.

Further exemplary magnetic particles for use with the solid support 10include those that have a magnetic core surrounded by a polymericmaterial. The polymeric material may be any polymeric material suitablefor use in assays such as polystyrene and polystyrene-divinyl benzene.In other embodiments, the magnetic particles may comprise chromiumdioxide magnetic particles (chrome particles). As will be discussedfurther below, these particles may have pendent surface groups such asamine functional groups which are aldehyde-reactive, or which can bemodified to include aldehyde-reactive groups.

Exemplary chromium oxide particles include those comprising a core ofchromium oxide that has a reduced surface, which is then coated withsilica and further coated with a silane as taught in U.S. Pat. No.4,661,408, the relevant disclosure of which is incorporated herein byreference. Other particular embodiments of magnetic and non-magneticparticles that may be employed are set forth in U.S. Pat. No. 6,231,982,the relevant disclosure of which is also incorporated herein byreference.

The solid support 10 may comprise or otherwise be modified so as toinclude surface functional groups 12 which are bound (by covalentbonding or otherwise) to the solid support 10, and which may bind to afirst binding pair member 16 directly or via a linking agent, e.g.,linking agent 14. In the latter case, the solid support 10 includessurface functional groups 12, which bind to one or more linking agents14. Each linking agent 14 may, in turn, bind to one or more firstspecific binding pair members 16. Exemplary suitable surface functionalgroups 12 include amine, hydrazine, hydrazide, aminooxy, cyanide,alcohol groups, and the like. In particular embodiments, the surfacefunctional groups 12 comprise amine functional groups.

In certain embodiments, as shown in FIG. 1, one or more linking agents14 may be utilized between the functional groups 12 on the solid support10 to immobilize one or more first binding pair members 16 on the solidsupport 10. In certain embodiments, the linking agent 14 comprises oneor more members from the group consisting of carbon, oxygen, sulfur,nitrogen, and phosphorous. In addition, the linking agent 14 may bealiphatic or aromatic. When heteroatoms are present, oxygen may bepresent as oxo or oxy, bonded to carbon, sulfur, nitrogen orphosphorous; nitrogen may be present as nitro, nitroso or amino, andbonded to carbon, oxygen, sulfur or phosphorous; sulfur may be analogousto oxygen; while phosphorous may be bonded to carbon, sulfur, oxygen ornitrogen, usually as phosphonate and phosphate mono- or diester. Incertain embodiments, any compound useful in forming a bond between thefirst binding pair member 16 and functional groups 12 on the solidsupport 10 may be utilized as the linking agent 14.

Additional exemplary linking agents 14 include but are not limited toaldehydes, dicarboxylic acids and anhydrides, polyamines, polyaldehydes,and heterobifunctional agents such as 2-iminothiolane hydrochloride,sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,m-maleimidosuccin-imide ester,N-succinimidyl-(4-iodoacetyl)aminobenzoate, and similar species known tothose skilled in the art.

Further linking agents 14 may include compounds comprising a nitrogengroup, a phosphate group; an amino group; an alkylating agent such ashalo or tosylalkyl, oxy (hydroxyl or the sulfur analog, mercapto);oxocarbonyl (e.g., aldehyde or ketone); or active olefin such as a vinylsulfone or α-, β-unsaturated ester. In an embodiment, these linkingagents 14 may immobilize the first binding pair member 16 on the solidsupport 10 by reaction with the surface functional groups 12 and thefirst binding pair member 16. Where an amine and a carboxylic acid orits nitrogen derivative or phosphoric acid are reacted, amides,amidines, and phosphoramides are formed. Where mercaptan and activatedolefin are linked, thioethers are formed. Where a mercaptan and analkylating agent are linked, thioethers are formed. Where an aldehydeand an amine are linked under reducing conditions, an alkylamine isformed. Where a ketone or aldehyde and a hydroxylamine (includingderivatives thereof where a substituent is in place of the hydrogen ofthe hydroxyl group) are linked, an oxime functionality (═N—O—) isformed. Where a carboxylic acid or phosphate acid and an alcohol arelinked, esters are formed. Various linking agents that may be utilizedherein are known in the art; see, for example, Cautrecasas, J. Biol.Chem. (1970) 245:3059.

In a particular embodiment, the linking agent 14 is one that will reactwith amine groups on the solid support 10 on the one hand and react withamine groups of the first binding pair member 16 on the other hand.Thus, in certain embodiments, the linking agent 14 may comprise analdehyde such as glutaraldehyde which includes at least two reactivecarbonyl groups—one of which will react with the functional groups 12 onthe solid support 10 and one which will react with the first bindingpair member 16.

The first specific binding pair member 16 may include any compound whichis selective for a target analyte in a sample, such as a correspondingsecond binding pair member 18. In an embodiment, the first binding pairmember 16 may thus comprise any member of a pair of components, whichmay bond or otherwise bind via an attractive force, fit, or the likewith at least a second member of the specific binding pair. In a certainembodiment, the first binding pair member 16 may bind to more than onesecond binding pair members 18. The first binding pair member 16 and theone or more second binding pair members 18 may each be complementarymembers of a specific binding pair as are known in the art such as:antigen-antibody; enzyme-substrate; polynucleotide interactions, and thelike. Exemplary binding pair members 16, 18 are set forth in U.S. Pat.No. 7,842,475, the entirety of which is hereby incorporated by referenceherein.

The sample 20 may be any suitable material suspected of having thetarget analyte, e.g., second binding pair member 18. In an embodiment,the target analyte may be any molecule found directly in a sample suchas biological tissue, including body fluids, from a suitable subject.The subject may be any human or non-human mammal such and the biologicalsample may comprise whole blood, serum, plasma, sputum, lymphatic fluid,semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool,cerebral spinal fluid, tears, mucus, and the like; biological tissuesuch as hair, skin, sections or excised tissues from organs or otherbody parts; and so forth. The sample 20 may undergo any pre-treatment orpreparation necessary to submit the sample to the solid support 10 aswould be appreciated by one skilled in the art.

Aspects of the present invention herein are directed to processes todetermine that sufficient binding sites exist for attachment of thefirst binding pair member 16. Thus, referring again to FIG. 1, it may becritical to know the number of surface functional groups 12 on the solidsupport 10 and/or the number of functional groups associated with thelinking agent 14 available for bonding to the first binding pair member16. During preparation of the solid support 10 for immobilizing firstbinding pair member 16 thereon, it may be useful to know the number ofsurface functional groups and/or linking agents that are immobilized onthe solid support 10. Further, prior to introduction of the firstbinding pair member 16 for immobilization on the support, knowledge ofthe number of binding sites available beforehand may assist withproviding an effective amount of the first binding pair member 16. Toreiterate, too few first binding pair members 16 for the number offunctional groups available for binding thereto may result in anunsaturated solid support 10 incapable of detecting the target analyte,especially at higher concentrations in the detectable range. Meanwhile,the processes described herein may prevent the addition of an excessiveamount of the binding pair member 16, thereby resulting in waste duringmanufacture of like solid supports.

Referring now to FIGS. 2-3, there are illustrated components utilized ina method for determining a number of functional groups on a solidsupport in accordance with an aspect of the present invention. Incertain embodiments, one or more of the components may be provided as akit and may further include packaging and/or instructions fixed in atangible form for carrying out a method as described herein.

In the embodiment of FIG. 2, there is shown a solid support 10 havingsurface functional groups 12 as was described above. In addition, aselective compound 22 is provided which may bind to, by covalent bondingor otherwise, to the surface functional groups 12 (shown as immobilizedselective compound 24). During this interaction between the selectivecompound 22 and the functional groups 12, an amount of the selectivecompound 22 may not bind to the surface functional groups 12. Thisamount will be “free” and being unbound to support 10 can be captured ina byproduct 26, such as a supernatant. As shown in FIG. 3, there isshown a byproduct 26 which comprises an amount of unbound selectivecompound 28. To this byproduct 26, an indicator 30 is provided which maybind to the unbound selective compound 28 to provide a measurableresult. The process for determining an amount of the functional groups12 utilizing the selective compound 22, 28 and indicator 30 will bedescribed in detail further below.

In another embodiment, as shown in FIG. 4, there is shown a solidsupport 10 having a linking agent 14 bonded to the solid support 10 viasurface functional groups 12. In this embodiment, the selective compound22 is thus one which may bind to, by covalent bonding or otherwise, tofunctional groups 15 of the linking agent 14 to provide immobilizedselective compound 24. Further, in this embodiment, during thecontacting between the selective compound 22 and the functional groups15, it is also appreciated that an amount of the selective compound 22may not bind to the surface functional groups 12. This amount will be“free” and may be captured in a byproduct 26, such as in a supernatant.Referring again to FIG. 3, the byproduct 26 can be collected and thuscomprises an amount of unbound selective compound 28. To this byproduct26, an indicator 30 is provided which may bind to the unbound selectivecompound 28 in the byproduct 26 to provide a measurable result. Theprocess for determining an amount of the functional groups 15 on thelinking agents 14 utilizing the selective compound 22, 28 and indicator30 will also be described in detail further below.

Referring now to FIG. 5, there is shown a method 100 for quantifying anamount of target functional groups on a solid support 10. It isunderstood that the term “target functional groups” may include anyfunctional groups directly or indirectly immobilized the solid support10, and thus may refer surface functional groups 12 or functional groups15 on a linking agent 14 as described above. For purposes of the method100, one set of target functional groups to quantify may be selected. Inan embodiment, the target functional groups are surface functionalgroups 12 on the solid support 10, which have been immobilized on thesolid support 10 by a reaction which adds the functional groups 12directly to the solid support 10. For example, immobilized aminofunctional groups may be provided by a reaction between an aminosilanecompound and the solid support 10. In other embodiments, the targetfunctional groups are present on the linking agent 14 and bind tosurface functional groups 12 on the solid support 10. In addition, thelinking agent 14 comprises target functional groups for binding with thefirst binding pair member 16. The target functional groups may compriseamino functional groups, aldehydic functional groups, or any otherfunctional groups described herein. The functional groups of the linkingagent 14 for binding to both the solid support 10 and the first bindingpair member 16 may be the same, but it is the understood that thepresent invention is not so limited.

In an embodiment, the method 100 comprises step 102 of contacting thetarget functional groups immobilized on the solid support 10 with aneffective amount of a selective compound 22. This contacting results inat least some of the selective compound 22 being bound to the solidsupport 10 via the target functional groups (12 or 15) and a byproduct26 (e.g., a supernatant) comprising unbound selective compound 22 (ifany). The contacting step 102 may take place at a suitable temperature,e.g., room temperature to 50° C., and for a suitable time period, e.g.,from 1 to 96 hours, for the binding to complete. Further, the contactingstep 102 may take place in a suitable vessel. In an embodiment, thecontacting comprises reacting the selective compound 22 and the targetfunctional groups (12 or 15).

In an embodiment, the method 100 may thus further include step 104 ofobtaining the byproduct 26, which may have an amount of the unboundselective compound 28. In an embodiment, the byproduct 26 may comprise asupernatant having an amount of the selective compound 22, which did notbind (covalently or otherwise) to the target functional groups (“unboundselective compound 28”). In a particular embodiment, the components maybe added to a vessel, placed in an incubator, and mixed for a suitableamount of time. Thereafter, a resulting byproduct 25 such as asupernatant may be separated from the solid support 10 by magneticseparation, centrifugation, or the like.

To determine the amount of the unbound selective compound 28, the method100 may further comprise step 106 of adding an indicator 30 to bind tothe unbound selective compound 28 in the byproduct 26 (e.g.,supernatant) to provide a measurable result. The addition of theindicator 30 may also take place at a suitable temperature, e.g., roomtemperature to 50° C., and for a suitable time period, e.g., from 1 to96 hours, effective to provide the measurable result. Withoutlimitation, the measurable result may comprise an absorbance value, afluorescence value, and an electrical current signal value. In certainembodiments, the measurable result may comprise a colorimetric result.

Accordingly, the indicator 30 may be any suitable compound to bringabout a measurable result. For example, the indicator 30 may comprise amember selected from the group consisting of propylamine,propionaldehyde, and 2, 4, 6-trinitrobenzene sulfonic acid (TNBSA) toprovide a colorimetric result. Moreover, individual ones of theseindicators may be more suitable than others for particular functionalgroups/selective compounds. For example, propionaldehyde may be asuitable indicator 30 when the unbound selective compound 28 comprisesone with one or more amino groups thereon which will react with thealdehydic selective compound. In turn, it is appreciated that theselective compound 22 may be any compound which binds at leasttemporarily to a target functional group and to which the indicator 30may bind. In this way, the indicator 30 may provide a measurable signalindicative of an amount of selective compound associated therewith.

Following addition of the indicator 30, the method 100 may furtherinclude step 108 of measuring the measurable result. The measurement maybe taken using suitable instrumentation known in the art such as amicroplate well reader which utilizes a selected detection mode. Commondetection modes include but are not limited to absorbance, fluorescenceintensity, luminescence, time-resolved fluorescence, and fluorescencepolarization. The detectors may be part of a system that also comprisesa computing unit comprising one or more modules configured to receivedata from the detectors and determine at least one result from the data.The system may also add any or all of the components for the methodsaccording to instructions provided by the computing unit. In this way,the computing unit may also function as an electronic control circuit.The computing unit may comprise, for example, a special purpose computercomprising a microprocessor, a microcomputer, an industrial controller,a programmable logic controller, a discrete logic circuit or othersuitable controlling device. In an embodiment, the computing unit mayfurther comprise one or more input channels, a memory, and outputchannel(s). The memory may include a computer-readable medium or astorage device, e.g., floppy disk, a compact disc read only memory(CD-ROM), or the like. In an embodiment, the computing unit may comprisecomputer readable instructions for performing any aspect of the methodsor for controlling any aspect of the components described herein.

In an embodiment, the method 100 further comprises step 110 ofdetermining from the measured result an amount of unbound selectivecompound 28 in the byproduct 26. The amount of the unbound selectivecompound 28 may be determined via the use of known standards andcontrols as would be well understood by persons skilled in the art. Forexample, results may be compared to values of a calibration curvecreated from a plurality of standard samples having predeterminedconcentrations as is known in the art.

Further, the method 100 may include step 112 of determining an amount ofimmobilized selective compound 24 immobilized on the solid support 10from the amount of unbound selective compound 28 in the byproduct 26,e.g., supernatant. The amount of immobilized selective compound 24 maybe determined via calculating a difference between a startingconcentration of selective compound 22 added for the contacting step 102and the determined amount of unbound selective compound 28 (from step110). The amount of immobilized selective compound 24 on the solidsupport 10 will be that amount bound (e.g., covalently bonded orotherwise) to the surface functional groups 12 or functional groups 15on the linking agents 14 depending on the particular design of theprocess.

Still further, the method may include step 114 of determining from theamount of the immobilized selective compound 24 an amount of the targetfunctional groups immobilized on the solid support 10. Thus, from theamount of immobilized selective compound 24, the corresponding number oftarget functional groups (e.g., functional groups 12 or 15) may bedetermined. For example, in certain embodiments, one immobilizedselective compound 24 may correspond to one target functional group onthe solid support 10. Alternatively, one immobilized selective compound24 may correspond to two or more functional groups on the solid support10. In certain embodiments, the number of target functional groups maybe expressed as the number of equivalents of the particular functionalgroups.

By way of example only, two specific embodiments will be explained inbrief detail below, although it is understood that the present inventionis not so limited.

In a first particular embodiment, the solid support 10 comprises surfacefunctional groups 12, e.g., immobilized aldehydic functional groups.Thus, in this instance, one objective of the method may be to determinethe number of aldehydic functional groups on the solid support 10. Toaccomplish this, the aldehydic functional groups may be immobilized onthe solid support 10 by a reaction which adds the aldehydic functionalgroups to existing amino groups on the solid support 10. In thisembodiment, the solid support 10 may comprise amino-functionalizedparamagnetic particles, which are readily commercially available. Inaddition, in a particular embodiment, the aldehydic functional groupsmay be immobilized on the solid support 10 via a reaction betweenglutaraldehyde and the amino groups on the solid support 10.

In a next step, a selective compound 22 which selectively reacts withthe immobilized aldehydic functional groups may be added to a vesselcomprising the solid support 10, mixed, and incubated for an effectiveamount of time. In this instance, the selective compound 22 may be onewhich readily reacts with the immobilized aldehydic functional groupssuch as an amino-containing compound. In an embodiment, the selectivecompound 22 comprises propylamine. After reaction between propylamineand the immobilized aldehydic functional groups, propylamine shouldideally react with substantially all to all of the available aldehydicfunctional groups provided sufficient propylamine was present. Thereaction may take place under suitable conditions effective to react thecomponents to completion and produce a supernatant. The remainingunbound propylamine may be disposed within a byproduct 26 such as asupernatant.

To this supernatant, an indicator 30 is added which, in this instance,will react with the unbound propylamine in the supernatant. In thisembodiment, the inventors have found that 2,4,6-trinitrobenzene sulfonicacid (TNBSA) works as a suitable indicator. TNBSA is a commerciallyavailable reagent used to quantitate amino groups. The reaction of TNBSAwith amines generates a highly chromogenic product that can be readilymeasured at a peak wavelength of 335 nm, or more conveniently at 405 nmwhen using a microplate. From the resulting measurements, the amount ofbound and unbound propylamine in this instance may be determined asdescribed above, and the number of aldehydic functional groups on thesolid support 10 may be determined therefrom.

In a second particular embodiment, the solid support 10 comprisesfunctional groups 12, e.g., immobilized amino functional groups. Thus,in this instance, one objective of the method is to determine the numberof amino functional groups on the solid support 10. To accomplish this,as mentioned above, PMP may be reacted with an am inosilane produceamino-functionalized PMP.

In a next step, a selective compound which selectively reacts with theimmobilized amino functional groups may be added to a vessel comprisingthe solid support 10, mixed, and incubated. In this instance, theselective compound 22 may be one which readily reacts with theimmobilized amino functional groups such as an aldehyde compound. In anembodiment, the selective compound comprises propionaldehyde. Afterreaction between propionaldehyde and the immobilized amino functionalgroups, propionaldehyde should ideally react with available aminofunctional groups provided sufficient propionaldehyde was present. As inthe above example, the reaction may take place under suitable conditionseffective to produce a supernatant. In this instance, remaining unboundpropionaldehyde may be disposed within the supernatant.

To this supernatant, an indicator 30 is added which, in this instance,will react with the unbound propylamine in the supernatant. In thisembodiment, the inventors have found that a solution comprising PURPALD®(4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole,4-Amino-5-hydrazino-1,2,4-triazole-3-thiol), available fromSigma-Aldrich (St. Louis, Mo.), works as a suitable indicator. TNBSA isa commercially available reagent used to quantitate amino groups. Thereaction of PURPALD® with carbonyl groups generates a highly chromogenicproduct. In an embodiment, the resulting product has a maximumabsorption at 490-540 nm. From the resulting measurements, the amount ofbound and unbound propionaldehyde may be determined as described above,and the number of immobilized amino functional groups on the solidsupport 10 may be determined therefrom.

In either case, following determination of the number of immobilizedfunctional groups, an effective amount of the first binding pair member16 may be reacted with the immobilized functional groups 24 on likesolid supports 10. In an embodiment, based on the determined number oftarget functional groups on the solid support 10, additional targetfunctional groups may be added to the solid support 10 if the determinednumber of target functional groups on the solid support 10 is less thana predetermined value. The predetermined value may be that number oftarget functional groups necessary to directly or indirectly bind apredetermined amount of the first binding pair members 16. In anotherembodiment, based on the determined number of target functional groupson the solid support 10, one or more additional solid supports may bemanufactured which comprise a stoichiometric amount of the first bindingpair member 16 bound to the target functional groups. As a result, thedetermined number of functional groups by the processes described hereinmay be utilized as a guide to determine optimum manufacturing of solidsupports.

In another aspect, a kit may be provided comprising any componentsnecessary for carrying out a method or process as described herein. Inan embodiment, the kit comprises the selective compound whichselectively binds to the target functional groups; and an indicatorwhich selectively binds to the selective compound as described herein.In another aspect, the kit may comprise packaging and/or instructionsfor carrying out a method as described herein. The instructions may beprinted or otherwise fixed, recorded, and/or saved on a tangible medium.

Aspects of the present invention are demonstrated by the followingexamples, which are not intended to be limiting in any manner.

Example 1 Determination of the Incubation Time Required for Quantitationof Amino Equivalences of Paramagnetic (PMP) Particles SamplePreparation:

Two lots of Siemens' amine-functionalized paramagnetic particles(AHI-PMP) were used for the time-dependence studies. To 800 mg of eachlot, 5×40 mL 25 mM sodium borate (pH 10.0, prepared from sodium borate,decahydrate, Sigma Cat. #S9640) was used to wash the particles in a50-mL Falcon Tube. Supernatant was removed after a magnetic separationusing DynaMag-50 (Life Technologies/Thermo Fisher) or an equivalent, andresuspended with 40 mL buffer after the last wash. 5 mL were equallyaliquoted into each of eight 15-mL Falcon tubes @ 100 mg each.

To each tube was added 1.6 mL of 50 mM propionaldehyde (Sigma) in water.The tubes were filled to 8 mL with 25 mM borate (pH 10.0). Tubes whichhad 100 mg PMP with 10 mM propionaldehyde were placed in a 50° C.incubator and mixed on an orbital mixer. At the following time points:1, 2, 3, 5, 15, 24, 48 and 72 hours, one tube each was removed andmagnetic separation was performed using DynaMag-15 or an equivalent. Thesupernatant was saved from each tube.

Zero to 2.5 mM propionaldehyde standard samples in water were preparedand ¼ dilutions of supernatant samples in water were prepared. Eachsample was pipeted into a 0.5 mL microfuge tube and an appropriate tubeholder was used so that an 8- or 12-multi-channel pipet could aspirate.When these preparations were completed, 7 mg/mL of PURPALD®(Sigma-Aldrich, St. Louis, Mo.) was dissolved in 1 M NaOH and vortexed.Thereafter, 1% volume of 0.3% hydrogen peroxide (ACROS Organics) wasmixed in. With a multi-channel pipet, 200 μL of thePURPALD®/NaOH/hydrogen peroxide solution was transferred into each of aplurality of wells, followed by 50 μL of standard samples andsupernatant samples. The samples were mixed on an orbital rotator mixerfor 10 to 30 minutes at room temperature. When the standard colorsappeared to be increasing strength by way of an increasing pink color,the microplate was moved to a microplate reader (e.g., MolecularDevices' Vmax kinetic microplate reader) and read at 490 nm.

Results:

As shown in FIG. 6, the average absorbance at 490 nm for 8 separatestandards (assayed in triplicate) were plotted and a quadratic fittingequation of μmole aldehyde vs. absorbance was established. The quadraticfitting equation was used to calculate the unbound μ moles ofpropionaldehyde, which was then used to calculate the μ moles ofaldehyde bound. The μ moles of aldehyde bound to the PMP were equivalentto the amino equivalence of amines on PMP, after a correction fromdilution factors.

In addition, as shown in FIG. 7, after 24 hours of PMP reaction withpropionaldehyde, Schiff base formation was completed indicating that thedetermination of the amino equivalences may be carried out at 50° C.incubator on a mixer for at least 24 hours.

Example 2 Determination of the Amino Group Equivalences of 12 Lots PMPSample Preparation

Twelve lots of PMP from Siemens Healthcare Diagnostics Inc. wereobtained. 100 mg of material from each lot was placed in a separate15-mL Falcon Tube and washed with 5×10 mL 25 mM sodium borate (pH 10.0).Supernatant was removed after magnetic separation using DynaMag-15 or anequivalent and then re-suspended with 5 mL buffer after the last wash.

To each tube was added 1.6 mL of 50 mM propionaldehyde (Sigma) in water.The tubes were filled to 8 mL with 25 mM borate (pH 10.0). The tubeswhich had 100 mg PMP with 10 mM propionaldehyde were placed in a 50° C.incubator and mixed on an orbital mixer for 72 hours. All tubes wereremoved and magnetic separation was performed using DynaMag-15 or anequivalent. The supernatant was saved from each tube.

Assay on a 96-Well Microplate

Zero to 2.5 mM propionaldehyde standard samples were prepared in waterand ¼ dilutions of supernatant samples were prepared in water. Eachsample (standards and supernatants) was pipeted into a 0.5 mL microfugetube and an appropriate tube holder was used so that an 8- or12-multichannel pipet could aspirate. Thereafter, 7 mg/mL of PURPALD®(catalog number 162892-25G, Sigma-Aldrich, St. Louis, Mo.) was dissolvedin 1 M NaOH and vortexed to dissolve completely. 1% volume of 0.3%hydrogen peroxide (ACROS Organics catalog number 426001000) was mixedinto the PURPALD® solution. With the multichannel pipet, 200 μL of thePURPALD®/NaOH/hydrogen peroxide was transferred into wells, followed by50 μL of standards and samples. Thereafter, the samples/standards weremixed on an orbital rotator mixer for 10 to 30 min at room temperature.When an increasing pink color appeared, the microplate was moved to amicroplate reader (e.g., Molecular Devices' Vmax kinetic microplatereader) and read at 490 nm.

Results

As shown in FIG. 8, the average absorbance of 8 standards (assayed intriplicates) were plotted and a quadratic fitting equation of μ molealdehyde vs. absorbance at 490 nm was established. The fitting equationwas used to calculate the unbound μmoles of propionaldehyde, which, inturn, was used to calculate the μmoles of propionaldehyde bound. The μmoles of propionaldehyde bound was equivalent to the amino equivalenceon the PMPs. The μ moles of amino equivalences per mg of PMP were thusdetermined. It was shown that the tested lots had about the same aminoequivalences per mg of solid.

Example 3 Determination of the Incubation Time Required for Quantitationof Aldehyde Equivalences of PMP-CHO Particles Sample Preparation:

Eight hundred mg of amino-functionalized PMP (Siemens HealthcareDiagnostics Inc.) were buffer exchanged with 0.1 M sodium phosphate (pH7.5) and activated by 6.25% glutaraldehyde (Polysciences Catalog #1909)at 50 mg solid/mL for 3 hours and mixed by a Glas-Col 3D orbital shaker(3D) for 3 hours to PMP-CHO particles. The unbound glutaraldehyde waswashed off by 4×56 mL with 0.1 M sodium phosphate (pH 7.5). ThePMP-CHO's were re-suspended in the new polypropylene bottles at 20 mgsolid/mL in the same buffer.

One hundred mg of the PMP-CHO were aspirated to each of eight 15mL-Falcon tubes, were washed by 5×10 mL of 25 mM sodium borate (pH 10,prepared from sodium borate, decahydrate, Sigma Catalog #S9640), andthen re-suspended in 5 mL of the same buffer. To each tube was added 1.6mL of 50 mM propylamine (Sigma cat #240958) in water and filled to 8 mLwith 25 mM sodium borate (pH 10), which resulted in the mixing of thesolid with 10 mM propylamine. The 8 tubes are incubated for 0-72 hoursin a 50° C. incubator. At each time point, the supernatant was separatedby a DynaMag-15 or an equivalent magnet and mixed with an equal volumeof 78 mM HEPES (Sigma catalog #H3375, pH not adjusted) to reach a finalpH of ˜8.5+/−0.2. The centrifuged supernatants were kept at 2-8° C.until all time points were completed.

Assay on a 96-Well Microplate:

Ten mL of mixed buffer was prepared by mixing 5 mL of 25 mM sodiumborate (pH 10), and equal volume of 0.5 M sodium bicarbonate (pH 7.9) toreach a pH of ˜8.8. 5 mM of propylamine standards were prepared bymixing equal volumes of 10 mM of propylamine and 78 mM HEPES, thendiluting to 0-5 mM propylamine in the mixed buffer. 200 μL of standardswere aspirated in triplicate into 96 microplate wells. In addition, 200μL of samples were aspirated in hexaplicate into 96 microplate wells. Toeach of standard and 3 of the sample wells was added 50 μL of 0.01%TNBSA (or Picrylsulfonic solution, Sigma cat #92823). 50 mM sodiumborate (pH 8.5). 50 μL of 50 mM sodium borate (pH 8.5) was added only tosample controls. The samples were mixed on a shaker at room temperaturefor ˜60 min. When the well solutions turned yellow to orange color toappropriate intensities (absorbance ˜0.5 to 1), the plate with wells wasmoved to a microplate reader such as Molecular Devices' Vmax kineticmicroplate reader, and read at 405 nm.

Results:

Table 2 below shows the A405 and μ moles aldehyde measured at each timepoint.

TABLE 2 Summary of the A405 and μ moles aldehyde/g PMP-CHO measured attime points Incub time u moles u moles hrs A405 BKG Net A405 unboundbound Dil factor mg PMP umole/mg 0 0.7095 0.0456 0.7083 1 0 80 100 0 1.50.6890 0.047 0.6423 0.8536 0.1464 80 100 0.117 3 0.6710 0.054 0.61730.8085 0.1915 80 100 0.153 16 0.6670 0.071 0.5963 0.7713 0.2287 80 1000.183 24 0.6630 0.076 0.5870 0.7550 0.2450 80 100 0.196 42 0.6610 0.0770.5843 0.7504 0.2496 80 100 0.200 48 0.6570 0.076 0.5810 0.7446 0.255480 100 0.204 72 0.6610 0.077 0.5843 0.7504 0.2496 80 100 0.200

FIG. 9 further shows the dependence of Schiff-base formation foraldehyde quantitation. In particular, the results indicated that at22+/2 hours incubation at 50° C., the reaction reaches completion.

Example 4 Determination of the Aldehyde Group Equivalences of PMP-CHOUsing Propylamine and Trinitrobenzene Sulfonic Acid (TNBSA) SamplePreparation:

Two grams of PMP (Siemens Healthcare Diagnostics Inc.) were bufferexchanged with 0.1 M sodium phosphate (pH 7.5) and activated by 6.25%glutaraldehyde (Polysciences Cat #1909) at 50 mg solid/mL for 3 hoursand mixed by a Glas-Col 3D orbital shaker (3D) or a Eberbach mixer (EB)at room temperature for 3 hours. The unbound glutaraldehyde was washedoff by 4×140 mL with 0.1 M sodium phosphate (pH 7.5). The PMP-CHO's werere-suspended in the new polypropylene bottles at 20 mg solid/mL in thesame buffer.

One hundred mgs in two 15 mL-Falcon tubes, labeled as 3D and EB, werewashed by 5×10 mL of 25 mM sodium borate (pH 10, prepared from sodiumborate, decahydrate, Sigma Cat #S9640) then re-suspended in 5 mL of thesame buffer. To each tube was added 1.6 mL of 50 mM propylamine (Sigmacat #240958) in water and the tubes were filled to 8 mL with 25 mMsodium borate (pH 10), which resulted in the mixing of the solid with 10mM propylamine. The two tubes were incubated for 24 hours in a 50° C.incubator. The supernatants were then separated by a DynaMag-15 or anequivalent magnet then mixed equal volume of 78 mM HEPES (Sigma catalog#H3375, pH not adjusted) to reach a final pH of ˜8.5+/−0.2. 10 mMpropylamine stock was prepared the same way as the samples except noPMP-CHO is present.

Assay on a 96-Well Microplate:

Ten mL of mixed buffer was prepared by mixing 5 mL of with equal volumeof 25 mM sodium borate (pH 10), and equal volume of 0.5 M sodiumbicarbonate (pH 7.9) to reach a pH of ˜8.8. 5 mM propylamine standardsamples were prepared by mixing equal volumes of propylamine and 78 mMHEPES first then diluted to 0-5 mM propylamine in the mixed buffer. 200μL of standard samples were aspirated in triplicate into 96 Microplatewells. 200 μL samples were aspirated in hexaplicate into 96 Microplatewells. To each standard sample and 3 sample wells were added 50 μL of0.01% TNBSA (or Picrylsulfonic solution, Sigma catalog #92823) in 50 mMsodium borate (pH 8.5) and 50 μL each of 50 mM sodium borate (pH 8.5)only to sample controls. The plate was mixed on a shaker at roomtemperature for ˜60 min. When the well solutions turned from yellow toorange color and reached to appropriate intensities (absorbance ˜0.5 to1), the plate was moved to a microplate reader, such as MolecularDevices' Vmax kinetic microplate reader, and read at 405 nm.

Results:

FIG. 10 shows μ moles of propylamine standards vs. A405. Table 3 belowshows the μ mole of aldehyde equivalences after subtracting the samplecontrols.

TABLE 3 Aldehyde equivalences of PMP-CHOs Sample # ABS umoles/mg PMP 3DCHO-PMP 0.485 0.1769 EB CHO-PMP 0.472 0.1951

Example 5 DOE Design and Determination of the Aldehyde GroupEquivalences of PMP-CHO Using Propylamine and TNBSA DOE Design:

DOE, Design of Experiments, version 17, a statistical method for designand analysis of experimental data is marketed by Minitab Inc. in StateCollege, Pa. A 3-factor full factorial design variable with a centerpoint and a response variable are listed in below:

TABLE 4 DOE Design and Response Variables Design Variables Low HighCenter PMP, mg/mL 7 70 38.5 Glutaraldehyde, % 1 12.5 6.75 VesselOccupancy, % 10  90 50 Response Variable μ molar aldehyde equivalents/mgPMP

Sample Preparation:

PMP ranged from 5 to 5.5 grams for 9 preparation conditions (2³+1=9) in1 L Nalgene square bottles. The PMP were washed and magneticallyseparated 3 times with wash buffer (10 mM sodium phosphate, pH 7.4).Supernatants were drained by pipet aspiration. To each wetcake, PMPcontaining residual buffer in various sizes of square Nalgene containers(0.25 to 8 L), is added to a calculated volume of diluted glutaraldehydeand mixed at room temperature for 3 hours via 3-D orbital shakersmanufactured by Glas-Col in Terre Haute, Ind.

After washing the PMP 2×, the PMP-CHO were transferred to new 1 LNalgene square containers and washed another 4 times with wash buffer.The final PMP-CHO wetcake is re-suspended in the 0.5 L Nalgene squarebottles with the wash buffer to reach 20 mg/mL and stored at 2-8° C.until assayed.

Assay on a 96-Well Microplate:

Prior to conducting assay on a microplate, 100 mg each of the sampleswere incubated with 10 mM propylamine in 25 mM sodium borate (pH 10) for24 hours at 50° C. using the assay protocol set forth in Example 5above.

Results:

Table 5 summarizes the sample conditions and the response variablevalues and

Table 5 A summary on DOE Design and Response Variable Values PMP Vesselumole StdOrder RunOrder CenterPt Blocks mg/mL GA % Occupancy CHO/mg 8 11 1 70 12.5 79 0.1165 6 2 1 1 70 1 79 0.0731 9 3 0 1 38.5 6.75 41.50.1059 4 4 1 1 70 12.5 4 0.138 1 5 1 1 7 1 4 0.0872 2 6 1 1 70 1 40.0785 3 7 1 1 7 12.5 4 0.1158 7 8 1 1 7 12.5 79 0.125 5 9 1 1 7 1 790.0879

Table 6 shows the fitting models from a statistical analysis. Inparticular, Table 6 shows the parameter values after fitting modelreduction with % Glutaraldehyde (p=0.011<0.05) the only factor that issignificant

TABLE 6 Analysis of Variance Source DF Adj. SS Adj. MS F-Value P-ValueModel 6 .003952 .000659 15.90 .060 Linear 3 .003601 .001200 28.97 .034PMP mg/mL 1 .000012 .000012 0.29 .644 GA % 1 .003553 .003553 85.76 .011Ves Occu 1 .000036 .000036 0.87 .449 2-way Interactions 2 .000342.000171 4.13 .195 PMP mg/mL*GA % 1 .000173 .000173 4.17 .178 PMPmg/mL*Ves Occu 1 .000169 .000169 4.09 .181 Curvature 1 .000009 .0000090.21 .690 Error 2 .000083 .000041 Total 8 .004035 Model Summary S R-sq(adj) R-sq (Pred) .0064368 97.95% 91.79%

The results are further shown in accompanying FIGS. 11-13.

FIG. 11 is an effect plot indicating % glutarahyde is a main factor ofaldehyde group formation.

FIG. 12 is an interaction plot showing PMP mg/mL intersecting with %Vessel Occupancy.

FIG. 13 is a Pareto Chart illustrating B (% Glutaraldehyde) is a maineffect of aldehyde group formation.

FIG. 14 illustrates that the Response Optimizer indicated that a D(Desirability) of 0.744 could be achieved with design variables set at70 mg/m L. 12.5% Glutaraldehyde, and 79% Vessel Occupancy.

In conclusion, the experiment of Example 5 indicated indicates that bycombining the Schiff-base formation between PMP-CHO and propylamine anda microplate test method with TNBSA, the optimization using DOE can beaccomplished.

While various embodiments of the present invention have been shown anddescribed herein, it will be obvious that such embodiments are providedby way of example only. Numerous variations, changes and substitutionsmay be made without departing from the invention herein. Accordingly, itis intended that the invention be limited only by the spirit and scopeof the appended claims.

The invention claimed is:
 1. A method for quantifying target functionalgroups immobilized on a solid support comprising one or moreparamagnetic particles, the method comprising the steps of: (a)contacting the target functional groups immobilized on the one or moremagnetic paramagnetic particles with an amount of a selective compound,wherein an amount of the selective compound binds directly to the targetfunctional groups and forms bound selective compound, and an amount ofthe selective compound does not bind to the target functional groups andis present as unbound selective compound, wherein the target functionalgroups comprise aldehydic functional groups, and wherein the selectivecompound comprises propylamine; (b) obtaining a byproduct having anamount of the unbound selective compound; (c) adding an indicator to thebyproduct which binds to unbound selective compound in the byproduct toprovide a colorimetric result, wherein the indicator comprises asolution comprising 2,4,6-trinitrobenzene sulfonic acid (TNBSA); (d)measuring the colorimetric result; (e) determining from the measuredresult an amount of unbound selective compound in the byproduct; (f)determining an amount of bound selective compound immobilized on the oneor more paramagnetic particles from the amount of unbound selectivecompound; and (g) determining from the amount of the immobilizedselective compound an amount of the target functional groups immobilizedon the one or more paramagnetic particles; and (h) reacting theparamagnetic particles with an effective amount of a first member of abinding pair to attach the first binding pair member to the paramagneticparticles, wherein the effective amount of the first binding pair memberis determined based upon the amount of target functional groupsimmobilized on the paramagnetic particles determined in step (g), andwherein the second binding pair member is a target analyte in a sample.2. The method of claim 1, wherein the aldehydic functional groups areimmobilized on the one or more paramagnetic particles by a reactionwhich adds the aldehydic functional groups to amino groups present onthe one or more paramagnetic particles.
 3. The method of claim 2,wherein in the aldehydic functional groups are immobilized on the one ormore paramagnetic particles via a reaction between glutaraldehyde andthe amino groups on the one or more paramagnetic particles.
 4. Themethod of claim 1, wherein the target functional groups are provided ona linking agent bonded to the one or more paramagnetic particles.
 5. Themethod of claim 1, further comprising, prior to step (h): manufacturingan additional one or more paramagnetic particles comprising additionaltarget functional groups immobilized on the one or more paramagneticparticles if the determined number of target functional groups on theone or more paramagnetic particles is less than a predetermined value.6. The method of claim 1, wherein the effective amount of the firstbinding pair member is a stoichiometric amount.
 7. The method of claim1, wherein the first binding pair member is an antigen, antibody,enzyme, substrate, or polynucleotide.
 8. The method of claim 1, whereinthe contacting the target functional groups with an amount of aselective compound comprises reacting the target functional groups withan amount of a selective compound.